Project 1 builds on advances of the previous funding cycle with the overall goal to elucidate HIV-1 determinants and cellular pathways responsible for deleterious effects of HIV-1 on astrocyte functions important for neuronal activity and survival. Among major findings, we demonstrated HIV-1 can replicate efficiently in astrocytes under certain conditions; identified a reservoir of HIV-1 DNA in brain astrocytes by Laser Capture Microdissection; began to dissect the mechanism of defective glutamate transport in infected astrocytes by cloning and characterization of promoters for glutamate transporters EAAT1 and (with collaborators) EAAT2 and by analysis of 3'-untranslated region (3'-UTR) of EAAT2 RNA, and began to investigate overall effects of HIV-1 on astrocyte biology through analysis of altered cellular transcripts by gene microarrays and subtractive hybridization. Our hypothesis is that HIV-1 exposure alters astrocyte gene expression for specific and definable impairments of cell functions essential to neuronal activity, including glutamate homeostasis. The proposed studies will further test this hypothesis with support of Cores and established interactions within the PO1, and collaborations outside. The Specific Aims are: 1) Using the EAAT1 and EAAT2 promoter clones and 3'-UTR constructs, compare the effect of HIV-1 on transcriptional and post-transcriptional control of glutamate transporter genes in astrocytes and on their function in preventing excitotoxic neuronal damage in human fetal brain cultures in vitro (in part with Project 2). 2) Using the services of U. Rochester Functional Genomics Center, continue to analyze cellular genes modulated by HIV-1 in astrocytes by gene microarrays, to construct an HIV-astrocyte custom microarray, and to use this database to investigate the potential role of HIV-1 strain variability in induction of gene expression in astrocytes impacting neuronal function. 3) Use the HIV-astrocyte microarray to investigate the response to HIV-1 in the context of physiological stimuli known to affect astrocyte gene expression and to employ gene silencing to pinpoint genes from the HIV-astrocyte microarray transducing the effects of HIV-1 upon EAAT1 and EAAT2 promoters. 4) Using a combination of neuronal functional assays employed by Project 4 and manipulations of gene expression in astrocytes, correlate HIV-1 induced changes in astrocyte biology indicated by microarray analyses with defects in signaling between astrocytes and neurons in compartmentalized cultures that permit assessment of changes in synaptic function and metabolism. 5) In collaboration with Project 3 and with the Neuropathology Core, test the correlation of selected HIV-1 induced changes in astrocyte biology indicated by gene microarray and functional analyses in Aims 2-4 to astrocyte functions in vivo that may contribute to neurodegeneration. These studies will further elucidate potential contributions of HIV-1 infection of astrocytes to neuropathogenesis and may lead to new ways of controlling the disease.